Grasses constitute about 20% of natural land cover (Kellogg, 2001) and the majority of cultivated biomass (57%) that can be sustainably produced in the U.S. (US-DOE, 2011). Rice straw, for example, represents approximately 23% of all agricultural waste, globally (Lal, 2005). The inefficiency of deconstructing cell walls into their component sugars represents a key limitation for production of biofuels from biomass via biological conversion (Lynd et al., 2008). On the other hand, root and leaf litter composition significantly affects soil carbon storage (Zhou et al., 2012). The cell wall components of whole grains also have beneficial effects in human health and various impacts on food processing (Fincher, 2009). For these reasons, grass cell wall properties critically impact unmanaged and managed ecosystems and economic uses.
In contrast to the cell walls of dicotyledenous plants (type I), cell walls of grasses and other Commelinoid monocots (type II) consist of up to 40% dry weight of the polysaccharide xylan, even in primary walls (reviewed in: (Carpita, 1996; Vogel, 2008; Scheller and Ulvskov, 2010)). Grass xylan is substituted with arabinofuranose side chains and infrequently with glucuronic acid (Obel et al., 2006). Of apparent significance to the structure of grass cell walls, ferulic acid (FA, FIG. 1A) esterifies to a fraction of the arabinose sidechains of xylan (reviewed in (Buanafina, 2009)). Dehydrodimers of ferulate (diferulates) form through oxidative coupling likely mediated by peroxidases (Takahama and Oniki, 1994; Bunzel et al., 2008) and cross-link adjacent xylan strands to one another (Ishii, 1991; Allerdings et al., 2005). Furthermore, the observation of ether linkages between ferulate and monolignols suggests that ferulic acid on arabinoxylan may nucleate lignin polymerization (Bunzel et al., 2004). Another hydroxycinnamate, para-coumaric acid (p-CA, FIG. 1A), is also ester-linked to grass cell walls. p-Coumaryl esters are more abundant on lignin, but have also been found to be esterified to grass arabinoxylan (Mueller-Harvey et al., 1986; Ishii et al., 1990; Faulds et al., 2004; Ralph, 2010). Though p-CA is readily oxidized to its radical, p-CA dimers have not been observed (Ralph et al., 1994). Rather p-coumaryl substituents may act as “radical catalysts” rapidly passing the radical to synapyl alcohols and facilitating lignin polymerization (Takahama and Oniki, 1994; Ralph, 2010).
FA on arabinoxylan, and especially diferulates, are thought to act to strengthen primary and secondary cell walls. For example, diferulate accumulation anticorrelates with fescue leaf elongation (MacAdam and Grabber, 2002). Similarly, hydroxycinnamate amounts anticorrelate with rice internode expansion (Sasayama et al., 2011). Cell wall FA content inversely correlates with enzymatic sugar release parameters in vitro (Grabber et al., 1998; Grabber et al., 1998; Lam et al., 2003; Casler and Jung, 2006). In addition, both cell wall-associated diferulates and free and cell wall-associated FA and p-CA deter fungal pathogens and insect pests of grasses (Santiago et al., 2007; Santiago et al., 2008; Lanoue et al., 2009).
Despite their importance, the proteins that incorporate hydroxycinnamates into grass cell walls are not well-characterized. Recently, Mitchell et al. (2007) proposed that a subclade of proteins with the Pfam domain, PF02458, for which transcripts are more abundant in grasses relative to dicots, might incorporate FA into grass walls. PF02458 domain-containing proteins are acyl CoA-dependent acyltransferases present in plants, fungi, and a few bacteria. In plants, these enzymes have been named BARD acyltransferase, based on the first biochemically characterized family members. They catalyze the addition of an acyl group from the thioester of coenzyme A primarily to oxygen nucleophiles of diverse acceptor molecules in plant secondary metabolism (reviewed in: (D'Auria, 2006)).
There are over 50 BARD members in most sequenced vascular plants (Table I). The BARD enzymes group robustly into five clades (D'Auria, 2006), though more recently subclades have been proposed (Tuominen et al., 2011). Several characterized members use hydroxycinnamoyl-CoAs as substrates, including the hydroxycinnamoyl-CoA:shikimate/quinate hydroxycinnamoyltransferase (HCT) involved in synthesis of lignin precursors (Hoffmann et al., 2003). Other recent reports have described suberin and cutin feruloyl transferases from Arabidopsis thaliana that act to transfer hydroxycinnamoyl-CoA ω-hydroxy fatty acids acyl acceptors (Molina et al., 2009; Rautengarten et al., 2012). Of importance for the possibility that the BAHD acyltransferase subclade identified by Mitchell, hereafter the “Mitchell clade”, might be involved in arabinoxylan modification, other BAHD enzymes catalyze the addition of esters to sugar acceptors, such as in anthocyanin biosynthesis (Unno et al., 2007). Indeed, Piston et al. found that rice plants simultaneously engineered with reduced expression of four genes from this clade show a ˜20% reduction in FA in young leaves (Piston et al., 2010). Furthermore, Withers et al. have recently described the biochemical characterization of one of the members of the “Mitchell clade”, PMT or here called OsAT4, which possesses p-coumaryl-CoA:monolignol aclytransferase activity (Withers et al., 2012).
There is a need to increase the digestibility of grass plants. This invention addresses that need.